Small peptides which inhibit binding to T-4 receptors and act as immunogens

ABSTRACT

Short peptide of the formula: 
     
         R.sup.a -Ser-Thr-Thr-Thr-Asn-Tyr-R.sup.b                   (I) 
    
     where R a  represents an amino terminal residue Ala- or D-Ala and R b  represents a carboxy terminal residue -Thr or -Thr amide or a derivative thereof with an additional Cys- residue at one or both of the amino and carboxy terminals, or a peptide of formula (II): 
     
         R.sup.1 -R.sup.2 -R.sup.3 -R.sup.4 -R.sup.5                (II) 
    
     where 
     R 1  is an amino terminal residue Thr-, Ser-, Asn-,Leu-,Ile-,Arg- or Glu- 
     R 2  is Thr, Ser or Asp 
     R 3  is Thr, Ser, Asn, Arg, Gln, Lys or Trp 
     R 4  is Tyr and 
     R 5  is a carboxy terminal amino group or a derivative thereof with a corresponding D- amino acid as the amino terminal residue, and/or a corresponding amide derivative at the carboxy terminal residue and/or additionally a Cys- residue at one or both of the amino and carboxy terminals, 
     or a physiologically acceptable salt thereof. 
     Such peptides bind to T4 receptors are useful in preventing viral infectivity by viruses which bind to the T4 receptors. These peptides are believed to act as competitive blocking agents.

This is a continuation of application Ser. No. 07/314,507, filed on Feb.15, 1989, now abandoned, which is a continuation of Ser. No. 07/048,148filed May 11, 1987 now abandoned, which is a continuation-in-part ofSer. No. 06/878,586 filed Jun. 26, 1986 now abandoned, which is acontinuation-in-part of Ser. No. 06/869,919 filed Jun. 3, 1986, nowabandoned.

BACKGROUND OF THE INVENTION

The complete nucleotide sequence of the AIDS (HIV) virus has beenreported by several investigators. (See Lee Ratner et al., Nature 313,p. 277, January 1985; Muesing et al., Nature 313, p. 450, February 1985;and Wain-Habson et al., Cell 40, pp. 9-17, January 1985.) The envelopegene has been associated particularly with antigenicity and infectivity.However, the envelope portion is also known to have regions which arehighly divergent. The HIV virus envelope glycoprotein has been shown toaffix covalently to the brain membranes of humans, rats, and monkeys andto cells of the immune system.

The realization that viruses may exert cell and tissue tropism byattachment at highly specific sites on cell membrane receptors hasencouraged investigators to seek agents which would bind at the viralreceptor sites of cell membranes and thus prevent binding of a specificvirus to these cells. A demonstration of specific receptor-mediatedvaccinia virus infectivity being blocked by synthetic peptides has beenpreviously demonstrated (Epstein et al., Nature 318: 663-667).

The HIV virus has been shown to bind to a surface molecule known as theCD4 or T4 region, which is present on various cells susceptable to HIVinfection, including T lymphocytes and macrophages. (See Shaw et al.,Science 226, pp. 1165-1171 for a discussion of tropism of HTLV-III.)

In addition to symptoms arising from immunodeficiency, patients withAIDS show neuropsychological defects. The central nervous and immunesystems share a large number of specific cell-surface recognitionmolecules, serving as receptors for neuropeptide-mediated intercellularcommunication. The neuropeptides and their receptors show profoundevolutionary stability, being highly conserved in largely unaltered formin unicellular organisms as well as higher animals. Furthermore, thecentral nervous and immune systems show common DC4 (T4) cell-surfacerecognition molecules which serve as receptors for the binding of HIVenvelope glycoprotein (gp 120). Since the same highly conservedneuropeptide informational substances integrate immune and brainfunction through receptors remarkably similar to those of HIV, wepostulated a very similar amino acid sequence between the HIVglycoprotein gp 120 and a short peptide previously identified in anothercontext from the envelope region of the Epstein Barr-Virus mightindicate the core peptide essential for viral receptor binding. It waspostulated that such a peptide would be useful in preventing infectionof cells with the HIV by binding with receptor cells and blocking thebinding of HIV gp 120, that such peptides binding to the receptor citeswould give rise to production of antibodies directed to the peptidesequence, and that these peptides might be used to provide immunologicalbasis for prevention of AIDS.

SUMMARY OF THE INVENTION

It is an object of the present invention to provide peptides which canalleviate symptoms of AIDS by preventing binding of HIV (AIDS virus)toreceptor sites of cells of brain membranes and the immune system.

It is also an object of the present invention to provide peptides foruse as vaccines to protect against development of AIDS in persons whomight become exposed to the HIV (AIDS virus).

It is a further object of the present invention to provide diagnosticmeans for identifying the presence of antibodies to HIV or HIV envelopeprotein.

An octapeptide in the HIV envelope glycoprotein (gp 120) was identifiedby computer-assisted analysis. This peptide, termed "peptide T" becauseof the high threonine content, has been shown to inhibit binding of gp120 to the brain membranes. The peptide has the sequenceAla-Ser-Thr-Thr-Thr-Asn-Tyr-Thr. Later analysis disclosed a class ofrelated pentapeptides having similar binding properties.

According to a first aspect of the present invention there is provided apeptide of formula (I):

    R.sup.a -Ser-Thr-Thr-Thr-Asn-Tyr-R.sup.b                   (I)

where R^(a) represents an amino terminal residue Ala- or D-Ala and R^(b)represents a carboxy terminal residue -Thr or -Thr amide or a derivativethereof with an additional Cys- residue at one or both of the amino andcarboxy terminals, or a peptide of formula (II):

    R.sup.1 -R.sup.2 -R.sup.3 -R.sup.4 -R.sup.5                (II)

where

R¹ is an amino terminal residue Thr-, Ser-, Asn-, Glu-, Arg-, Ile- orLeu-,

R² is Thr, Ser or Asp,

R³ is Thr, Ser, Asn, Arg, Gln, Lys or Trp

R⁴ is Tyr and

R⁵ is preferably a carboxy terminal residue -Thr, -Arg or -Gly or aderivative thereof with a corresponding D- amino acid as the aminoterminal residue, and/or a corresponding amide derivative at the carboxyterminal residue and/or additionally a Cys- residue at one or both ofthe amino and carboxy terminals.

While the preferred amino acids at R⁵ have been designated, it is knownthe amino acid at this position may vary widely. In fact, it is possibleto terminate the peptide with R⁴ (Tyrosine) as the carboxy terminalamino acid wherein R⁵ is absent. Such peptides retain the bindingproperties of the group taught herein. Serine and threonine appear to beinterchangeable for purposes of biological properties taught herein. Theactive compounds of the invention may exist as physiologicallyacceptable salts of the peptides.

This class of peptides has been found to bind to the T4 viral receptors.

Most preferred peptides, as well as peptide T above, are the followingoctapeptides of formula (I):

    D-Ala-Ser-Thr-Thr-Thr-Asn-Tyr-Thr

    and

    D-Ala-Ser-Thr-Thr-Thr-Asn-Tyr-Thr-amide

and the following pentapeptides of formula (II):

    Thr-Asp-Asn-Tyr-Thr

    Thr-Thr-Ser-Tyr-Thr

    Thr-Thr-Asn-Tyr-Thr

and their analogues with D-Thr as the amino terminal residue and/or anamide derivate at the carboxy terminal.

The compounds of the invention may be beneficially modified by methodsknown to enhance passage of molecules across the blood-brain barrier.Acetylation has proven to be especially useful for enhancing bindingactivity of the peptide. The terminal amino and carboxy sites areparticularly preferred sites for modification.

The peptides of this may also be modified in a constraining conformationto provide improved stability and oral availability.

The following abbreviations are used hereinafter:

    ______________________________________                                        Amino Acid  Three Letter Code                                                                           One Letter Code                                     ______________________________________                                        arginine    arg           R                                                   asparagine  asn           N                                                   aspartic acid                                                                             asp           D                                                   cysteine    cys           C                                                   glycine     gly           G                                                   serine      ser           S                                                   threonine   thr           T                                                   tyrosine    tyr           Y                                                   ______________________________________                                    

Unless otherwise indicated the amino acids are, of course, in thenatural form of L-stereoisomers.

A comparison of amino acid sequences of 12 pentapeptides is presented inTable 1. Although an initial computer search revealed peptide T(contained in the ARV isolate) to be the relevant moiety, as additionalviral sequences became available it become clear that the relevant,bioactive sequence, was a shorter pentapeptide comprising, nominally,peptide T[4-8], or the sequence TTNYT. In the isolates we compared(Table 1) substantial homologies were discerned only in this, shorter,region. The majority of changes are the interconversions of serine (S)and threonine (T), two closely related amino acids. The tyrosine ofposition 7 of peptide T is an invariant feature of all these constructsindicating that it may be obligatory for bioactivity. Substitutionsoccurring at position 5 include T, G, R or S. Position 4 and 6 werefirst restricted (with one exception) to S, T and N, all amino acidscontaining uncharged polar groups with closely similar stericproperties. An assessment of general sequence concordance among 5various AIDS viral isolates reveals that the region around and includingthe peptide T sequence is a highly variable area. Such variability mayindicate specialization through strong selective diversification of thefunction(s) which may be defined at this locus. Like the opiatepeptides, these peptide T analogs seem to exist in multiple forms,reminiscent of met and leu enkephalin. These pentapeptide sequencesrepresented in these various AIDS virus isolates are biologically activeand capable of interacting as agonists of the CD4 receptor--previouslyknown largely as a surface "marker" of T helper cells.

                  TABLE 1                                                         ______________________________________                                        Comparison of ENV Sequence from Multiple                                      AIDS Virus Isolates                                                           Isolate      Sequence     Reference                                           ______________________________________                                        peptide T    ASTTTNYT     Pert, C. B. et al.                                                            PNAS (in press)                                     .sup.1 ARV (195-199)                                                                       TTNYT        Willey, R. L. et al.                                LAV          TTSYT        PNAS 83: 5038, 1986                                 Z3           SSTYR                                                            NY5          NTSYT                                                            B10 (HTLV-III)                                                                             TTSYT        Starcich, B. R. et al.                              WMJ-1        SSTYR        Cell 45: 637, 1986                                  HAT-3        NTSYG                                                            Sequential isolates                                                                        STNYR                                                            WMJ-1        SSTYR        Hahn, B. L. et al.                                  WMJ-2        SSRYR        Science 232: 1548,                                  WMJ-3        SSTYR        1986                                                             TTSYS                                                            ______________________________________                                         .sup.1 Numbers refer to relative positions of amino acids within the ARV      env sequence.                                                            

The seven amino acid peptide CYS-THR-THR-ASN-TYR-THR-CYS is also active.Addition of cysteines to a core does not adversely affect activity.

The peptides were custom synthesized by Peninsula laboratories under aconfidentiality agreement between the inventors and the manufacturer.The Merrifield method of solid phase peptide synthesis was used. (SeeU.S. Pat. No. 3,531,258 which is incorporated herein by reference.) Thesynthesized peptides are especially preferred. While peptide T and thepentapeptide which is a portion thereof could be isolated from thevirus, the peptides prepared in accord with Merrifield are free of viraland cellular debris. Hence, (untoward reactions to contaminants does notoccur when the synthesized peptides are used.

The peptides of the invention may be produced by conventional methods ofpeptide synthesis. Both solid phase and liquid phase methods may beused. We have found the solid phase method of Merrifield to beparticularly convenient. In this process the peptide is synthesized in astepwise manner while the carboxy end of the chain is covalentlyattached to the insoluble support. During the intermediate syntheticstages the peptide remains in the solid phase and therefore can beconveniently manipulated. The solid support is a chloromethylatedstyrene-divinylbenzene copolymer.

An N-protected form of the carboxy terminal amino acid, e.g. at-butoxycarbonyl protected (Boc-) amino acid, is reacted with thechloromethyl residue of the chloromethylated styrene divinylbenzenecopolymer resin to produce a protected amino acyl derivative of theresin, where the amino acid is coupled to the resin as a benzyl ester.This is deprotected and reacted with a protected form of the nextrequired amino acid thus producing a protected dipeptide attached to theresin. The amino acid will generally be used in activated form, e.g. byuse of a carbodiimide or active ester. This sequence is repeated and thepeptide chain grows one residue at a time by condensation at the aminoend with the required N-protected amino acids until the required peptidehas been assembled on the resin. The peptide-resin is then treated withanhydrous hydrofluoric acid to cleave the ester linking the assembledpeptide to the resin, in order to liberate the required peptide. Sidechain functional groups of amino acids which must be blocked during thesynthetic procedure, using conventional methods, may also besimultaneously removed. Synthesis of a peptide with an amide group onits carboxy terminal can be carried out in conventional manner, using a4-methylbenzhydrylamine resin.

The compounds of the invention were found to effectively block receptorsites of cells and to prevent cell infectivity with HIV (AIDS virus) inmonkey, rat, and human brain membranes and cells of the immune system.

Pharmaceutical compositions according to the present invention comprisea peptide of the invention in association with a pharmaceuticallyacceptable carrier or excipient, adapted for use in human or veterinarymedicine. Such compositions may be presented for use in conventionalmanner in admixture with one or more physiologically acceptable carriersof excipients. The compositions may optionally further contain one ormore other therapeutic agents which may, if desired, be a differentantiviral agent.

Thus, the peptides according to the invention may be formulated fororal, buccal, parenteral, topical or rectal administration.

In particular, the peptides according to the invention may be formulatedfor injection or for infusion and may be presented in unit dose form inampoules or in multidose containers with an added preservative. Thecompositions may take such forms as suspensions, solutions, or emulsionsin oily or aqueous vehicles, and may contain formulatory agents such assuspending, stabilizing and/or dispersing agents. Alternatively, theactive ingredient may be in powder form for constitution with a suitablevehicle, e.g. sterile, pyrogen-free water, before use.

The pharmaceutical compositions according to the invention may alsocontain other active ingredients such as antimicrobial agents, orpreservatives.

The compositions may contain from 0.001-99% of the active material.

The invention further provides a process for preparing a pharmaceuticalcomposition which comprises bringing a peptide of the invention intoassociation with a pharmaceutically acceptable excipient or carrier.

For administration by injection or infusion, the daily dosage asemployed for treatment of an adult human of approximately 70 kg bodyweight will range from 0.2 mg to 10 mg, preferably 0.5 to 5 mg, whichmay be administered in 1 to 4 doses, for example, depending on the routeof administration and the condition of the patient.

It was postulated that the affinity constants are similar to those ofmorphine. On the basis of this affinity, dosage of 0.33-0.0003 mg/kg perday was suggested. This has proven to be effective. A bloodconcentration 10⁻⁶ to 10⁻¹¹ molar blood concentration is suggested. Inmonkeys 3 mg/kg per day achieves a serum concentration of 150×10⁻⁹ M.This concentration is 15 times greater than necessary to achieve aconcentration of 10⁻⁸ M. Primates generally require 10 times the doseused in humans.

A further aspect of this invention relates to vaccine preparationscontaining a peptide according to the invention, to provide protectionagainst infection by AIDS virus. The vaccine contains an effectiveimmunogenic amount of peptide, e.g. 1 μg to 20 mg/kg of host, optionallyconjugated to a protein such as human serum albumin, in a suitablevehicle, e.g. sterile water, saline or buffered saline. Adjuvants may beemployed, such as aluminum hydroxide gel. Administration may be byinjection, e.g. intramuscularly, interperitoneally, subcutaneously orintravenously. Administration may take place once or at a plurality oftimes, e.g. at 1-4 week intervals.

Antigenic sequences from crab as well as proteins from otherinvertebrates can also be added to the peptides of the invention topromote antigenicity.

A yet further aspect of this invention relates to test kits for thedetection of the AIDS virus and antibodies to the AIDS virus containinga peptide according to the invention as source of antigen, or amonoclonal antibody elicited by a peptide according to the invention.For example, a peptide according to the invention may be used in a testkit to detect AIDS infection and to diagnose AIDS and pre-AIDSconditions by using it as the test reagent in an enzyme-linkedimmunosorbent assay (ELISA) or an enzyme immunodot assay. Such test kitsmay include an insoluble porous surface or solid substrate to which theantigenic peptide or monoclonal antibody has been preabsorbed orcovalently bound, such surface or substrate preferably in the form ofmicrotiter plates or wells; test sera; heteroantisera which specificallybind to and saturate the antigen or antibody absorbed to the surface orsupport; various diluents and buffers; labelled conjugates for thedetection of specifically bound antibodies and other signal-generatingreagents such as enzyme substrates, cofactors and chromogens.

The peptide according to the invention may be used as an immunogen toelicit monoclonal antibodies which specifically bind to the relevantportion of the envelope sequence of the AIDS virus, using conventionaltechniques; such monoclonal antibodies form a further feature of theinvention.

DETAILED DESCRIPTION OF THE INVENTION Radiolabeling of gp 120,Preparation of Brain Membranes, Binding and Crosslinking of gp120 toReceptor, and Immunoprecipitation of T4 Antigen

HTLV-IIIB isolate of HIV was propaged in H9 cells, and the gp120 wasisolated by immunoaffinity chromatography and preparative NaDodSO₄/PAGE. Purified gp120 was labeled with ¹²⁵ I by the chloramine-T method.

Fresh human, monkey, and rat hippocampus were quickly homogenized(POLYTRON, Brinkmann Instruments) in 100 vol of ice-cold 50 Mm Hepes (pH7.4). The membranes collected by centrifugation (15,000 ×g) were washedin the original buffer volume and were used fresh or stored at -70° C.Before use, brain membranes and highly purified T cells (ref. 16; giftof Larry Wahl) were preincubated for 15-30 min in phosphate-bufferedsaline (PBS). Membranes derived from 2 mg (initial wet weight) of brain(≈100 μg of protein) were incubated with 28,000 cpm of ¹²⁵ I-gp120 for 1hr at 37° C. in 200 μl (final volume) of 50 mM Hepes containing 0.1%bovine serum albumin and the peptidase inhibitors bacitracin (0.005%),aprotinin (0.005%), leupeptin (0.001%), and chymostatin (0.001%).Incubations were rapidly vacuum-filtered and counted to determine thereceptor-bound material.

Immunoprecipitation

Immunoprepipitates were prepared by incubation (overnight at 4° C.) of0.5%- TRITON X-100/PBS-solubilized, lactoperoxidase/glucose oxidase/¹²⁵I-iodinated brain membranes or intact T cells with indicated mAbs at 10μg per reaction. A solid-phase immunoabsorbant (immunobeads, Bio-Rad)was used to precipitate immune complexes prior to their resolution byNaDodSO₄ /PAGE. Control incubations contained no primary mAb or asubclass control mAb (OKT8).

Chemical Neuroanatomy and Computer-Assisted Densitometry

Cryostat-cut 25-μm sections of fresh-frozen human, monkey, and rat brainwere thaw-mounted and dried onto gelatin-coated slides, and receptorswere visualized. Incubations, with or without antibodies (10 μg/ml)against T4, T4A, T8, and T11, were conducted overnight at O° C. in RPMImedium, crosslinked onto their antigens, and visualized with ¹²⁵I-labeled goat anti-mouse antibody. Incubations of slide-mounted tissuesections to label the antigen-receptor with ¹²⁵ I-gp120 were conductedin 5-ml slide carriers with (1 μM) or without unlabeled gp120 or nAbOKT4A (10 μg/ml) (Ortho Diagnostics).

Separation of T-Lymphocyte Subsets

Subsets of T cells were obtained by treatment of PERCOLLdensity-purified peripheral blood T cells with specific monoclonalantibodies (T4or T8) at 10 μg/ml. The treated cells were then panned(21) on a plastic Petri dish that was coated with goat [F(ab')₂ ]antimouse immunoglobulin (Sero Lab, Eastbury, Mass.) for 30 min at 4° C.The nonadherent cells were then removed, washed, and analyzed forreactivity by flow cytometry. The separated T4 and T8 cell populationshave<5% contamination of other T-cell subsets. Cells were then culturedwith phytohemagglutinin (1 μg/ml) for 72 hr and exposed to HIV asdescribed below. Infected cells were phenotypically characterized whencytotoxicity assays were performed.

Virus Infection

The HTLV-III virus used for infection was isolated from an interleukin 2(IL-2)-dependent cultured T-cell line established from fresh AIDSpatient material and passaged into HuT 78, a permissive IL-2-independentcell line.

DESCRIPTION OF THE DRAWINGS

FIG. 1A shows a crosslinking of ¹²⁵ I-gp120 to brain membranes and Tcells (a) ¹²⁵ I-gp120 only; (b) monkey; (c) rat; (d) human brain; and(e) human T cells.

FIGS. 1B and 1C show immunoprecipitation of ¹²⁵ I-labeled monkey brainmembranes and human T cells, respectively; (f,i) no primary antibodycontrol; (g,j) OKT4 Mab; (h,k) OKT8 Mab.

FIG. 2A shows a displacement of specific ¹²⁵ I-gp120 binding to freshrat hippocampal membranes. Each determination was performed intriplicate; the results of one experiment, which was performed threetimes with similar results, is shown.

FIG. 2B shows specific binding displaceable by 0.1 μg/ml of OKT4 and 4Aranged between 27 and 85% of total binding, which was 2,201+74 cpm inthe experiment shown.

FIG. 2C shows that peptide T and two of its synthetic analogssignificantly inhibited ¹²⁵ I-gp-120 binding in the 0.1 nM range.

FIG. 3 shows that viral infectivity is blocked by peptide T and itssynthetic analogs. Each determination was performed in duplicate.Results represent a single experiment which was repeated three timeswith similar results.

FIG. 3 shows that substitution of D-Ala or L-Ala results in aconsistently more potent analog than peptide T, and that amidation ofthe C terminal of threonine consistently produces greater potency.

EXAMPLE I

A single radiolabeled crosslinking product of about 180 Kd was obtainedafter specific binding of ¹²⁵ I-gp120 to membranes from either squirrelmonkey, rat or human brain membranes which are indistinguishable fromthat of human T cells (FIG. 1A). This result indicates that gp120 can becoupled to an approximately 60 Kd protein; unreacted ¹²⁵ I-gp120 runsadjacent to the no membrane control (lane a).

Immunoprecipitation of radioiodinated human brain membranes with OKT4and OKT8 (10 μg/ml) (FIG. 1B) shows that brain membranes contain a T4antigen of about 60 Kd, indistinguishable from that identified on humanT lymphocytes (FIG. 1C); by contrast, OKT8 immunoprecipitates a low(about 30 Kd) molecular weight protein from T lymphocytes (FIG. 1C)which is absent in brain membranes (FIG. 1B) indicating that brain T4 isnot derived from resident lymphocytes. Similar results were observedwith monkey and rat (not shown) hippocampal membranes. These resultsshow that the T4 antigen serves as the viral receptor and is a highlyconserved 60 Kd molecule shared by the immune and central nervoussystems.

The realization that Epstein-Barr and HTLV-III/LAV share an almostidentical octapeptide sequence caused the synthesis and study of"peptide T." FIG. 2 demonstrates the high (0.1 nM range) affinity andsaturability (FIG. 2A) of ¹²⁵ I-gp120 binding to freshly prepared ratbrain membranes. Specificity (FIG. 2B) is demonstrated by blockade withOKT4 and OKT4A, but not OKT3 (0.1 μg/ml). Peptide T and two of itssynthetic analogs (but not the irrelevant octapeptide substance P [1-8])significantly inhibited ¹²⁵ I-gp120 binding in the 0.1 nM range (FIG.2C). Substitution of a D-threonine-amide in position 8 resulted in atleast a 100-fold loss of receptor binding activity. The classical[D-Ala] substitution for [L-Ala] results in a consistently more potent,presumably more peptidase-resistant, analog than peptide T; amidation ofthe C terminal threonine also consistently produces somewhat greaterpotency (FIG. 3).

When the synthetic peptides were tested for their ability to block viralinfection of human T cells, experimentors were blind to binding assayresults. At 10⁻⁷ M the three peptides active in the binding assayreduced detectable levels of reverse transcriptase activity by almost9-fold. The less active binding displacer [D-Thr]-peptide T similarlyshowed greatly reduced blockade of viral infection, requiringconcentrations 100-fold higher to achieve significant inhibition. Thus,not only the rank order of potencies of the four peptides (D-[Ala]₁-peptide T-amide>D-[Ala]₁ -peptide T>peptide T>D-[Thr]₈ -peptideT-amide), but also their absolute concentrations in inhibiting receptorbinding and viral infectivity are closely correlated (FIG. 3).

EXAMPLE 2

An approximate 60-Kd protein, which is similar if not identical to humanT cell T4 antigen, was present in apparently conserved molecular form onmembranes prepared from human brain; furthermore, the radiolabeled HIVenvelope glycoprotein (¹²⁵ I-gp120) can be covalently crosslinked to amolecule present in three mammalian brains whose size andimmunoprecipitation properties were indistinguishable from the T4antigen. Using a method for visualizing antibody-bound receptors onbrain slices, the neuroanatomical distribution pattern of brain T4,which is densest over cortical neuropil and analogously organized in allthree mammalian brains, was presented. Also, radiolabeled HIV viralenvelope glycoprotein bound in an identical pattern on adjacent brainsections, once again suggesting that T4 was the HIV receptor.

EXAMPLE 3

Chemical Neuroanatomy, Computer-Assisted Densitometry. Cryostat-cut 25micron sections of fresh-frozen human, monkey, and rat brain werethaw-mounted and dried onto gel-coated slides and receptors visualizedas described by Herkenham and Pert, J. Neurosci., 2: 1129-1149 (1982).Incubations, with or without antibodies (10 μg/ml) against T4, T4A, T8and T11, were conducted overnight at 0° C. in RPMI, crosslinked ontotheir antigens and visualized with ¹²⁵ I-goat anti-mouse antibody.Incubations of slide-mounted tissue sections in order to labelantigen/receptor with ¹²⁵ I-gp120 were conducted in 5 ml slide carrierswith (10⁻⁶ M) or without unlabeled gp120 or Mab OKT4A (10 μg/ml) (OrthoDiagnostics) as described above for membranes.

Computer-assisted transformation of autoradiographic film opacity intoquantitative color images was performed. Co-exposure of standards ofknown increments of radioactivity with the monkey brain sectionsgenerated a linear plot (4=>0.99) of log O.D. versus cpm from which therelative concentration of radioactivity can be meaningfullyextrapolated. Cell staining of brain sections with thionine wasperformed by classical methods and visualization of receptors overlyingstained tissue.

EXAMPLE 4

Experiments were conducted to determine the distribution of T4 antigenon a rostral to caudal series or coronal sections of squirrel monkeybrain. These experiments demonstrated that there are detectable levelsof T4 monoclonal antibody binding to cytoarchitectonically meaningfulareas of the brain stem (e.g., the substantia nigra), but the strikingpattern of cortical enrichment is apparent at every level of theneuroaxis. OKT8, a T-lymphocyte directed monoclonal antibody from thesame subclass as OKT4, exhibits no observable pattern. Generally, themore superficial layers within the cerebral cortex contain the densestconcentrations of the T4 antigen; the frontal and perilimbic cortexoverlying the amydala are particularly receptor-rich throughout the deeplayers. The hippocampal formation has the densest concentration ofreceptors in the monkey, rat, and human brain. Dark field microscopy ofsquirrel monkey sections dipped in photographic emulsion revealed thatthe band of densest receptor labelling is located within the molecularlayers of the dentate gyrus and hippocampus proper (which contain veryfew neurons). Thus, receptors appear to be rightly distributed over theneuropil (the neuronal extensions of dendrites and axons) or may belocalized to a specific subset of unstained astroglial cells.

Evidence of the specificity of the chemical neuroanatomy and resultsshowing that T4 and the viral envelope recognition molecule areindistinguishable has been determined. Coronal sections of rat brainrevealed a very similar cortex/hippocampus-rich pattern of receptordistribution whether OKT4 or ¹²⁵ I-gp120 was used for visualization.Furthermore, this pattern was not apparent when incubation occurred inthe presence of unlabeled gp120 (]1 μM), OKT4A (10 μg/ml) or OKT4 (10μg/ml). Other mouse Mabs directed against other human T cell surfaceantigens including OKT8 and OKT11 gave no detectable pattern on ratbrain when visualized by ¹²⁵ I-goat anti-mouse IgG secondary antibodyjust as there was no reproducible, detectable antigen/receptor withsecondary antibody alone.

What is claimed:
 1. A peptide selected from peptides of the formula:

    Ra-Ser-Thr-Thr-Thr-Asn-Tyr-Rb

    or

    R.sup.1 -R.sup.2 -R.sup.3 -R.sup.4 -R.sup.5

wherein R_(a) is selected from the group consisting of amino terminalresidue Ala- and D-Ala; Rb is selected from the group consisting ofcarboxy terminal residue -Thr, -Thr amide and -Thr-Cys, R¹ is Thr-; R²is selected from the group consisting of Thr and Asp; R³ is selectedfrom the group consisting of Thr, Ser and Asn; R⁴ is Tyr; R⁵ is selectedfrom the group consisting of carboxy terminal residue -Thr, -Thr amidesand -Thr-Cys.
 2. A peptide according to claim 1 selected from the groupconsisting of:

    D-Ala-Ser-Thr-Thr-Thr-Asn-Tyr-Thr;

    and

    D-Ala-Ser-Thr-Thr-Thr-Asn-Tyr-Thr-amide.


3. A pentapeptide selected from the group consisting of:

    Thr-Asp-Asn-Tyr-Thr;

    Thr-Thr-Ser-Tyr-Thr;

    Thr-Thr-Asn-Tyr-Thr

and amides thereof.
 4. A peptide according to claim 1 of the formulaAla-Ser-Thr-Thr-Thr-Asn-Tyr-Thr.
 5. A peptide consisting of the aminoacid sequence Cys-Thr-Thr-Asn-Tyr-Thr-Cys.
 6. A pharmaceuticalcomposition comprising an effective amount of at least one peptide ofclaim 1 in a pharmaceutically acceptable carrier.
 7. A pharmaceuticalcomposition comprising an effective amount of at least one peptide ofclaim 2 in a pharmaceutically acceptable carrier.
 8. A pharmaceuticalcomposition comprising an effective amount of at least one peptide ofclaim 3 in a pharmaceutically acceptable carrier.
 9. A pharmaceuticalcomposition comprising an effective amount of at least one peptide ofclaim 4 in a pharmaceutically acceptable carrier.
 10. A pharmaceuticalcomposition comprising an effective amount of at least one peptide ofclaim 5 in a pharmaceutically acceptable carrier.